1

IWantAHighMCATScore’s
Guide to MCAT Lab Techniques
 2

Contents
Gel Electrophoresis ........................................................................................................................................ 3
Native-PAGE .............................................................................................................................................. 3
SDS-PAGE .................................................................................................................................................. 3
Reducing SDS-PAGE ................................................................................................................................... 4
Isoelectric Focusing .................................................................................................................................... 4
Western Blotting (Protein) ............................................................................................................................. 4
Southern (DNA) and Northern (RNA) Blotting .................................................................................................. 5
DNA Sequencing (Sanger Dideoxynucleotide Sequencing) ................................................................................ 6
Chromatography............................................................................................................................................ 7
Liquid Chromatography .............................................................................................................................. 7
High-Performance Liquid Chromatography (HPLC) ....................................................................................... 7
Gas Chromatography ................................................................................................................................. 7
Gel-Filtration (Size Exclusion) Chromatography............................................................................................ 7
Ion-Exchange Chromatography ................................................................................................................... 8
Affinity Chromatography ............................................................................................................................ 8
Thin-Layer Chromatography ....................................................................................................................... 8
Distillation .................................................................................................................................................... 9
Simple Distillation ...................................................................................................................................... 9
Fractional Distillation ................................................................................................................................. 9
Vacuum Distillation .................................................................................................................................... 9
Polymerase Chain Reaction ............................................................................................................................ 9
Spectroscopy ............................................................................................................................................... 10
1
H-NMR Spectroscopy .............................................................................................................................. 10
13
C-NMR spectroscopy ............................................................................................................................. 11
IR Spectroscopy ....................................................................................................................................... 11
UV-Vis Spectroscopy ................................................................................................................................ 11
Autoradiography ......................................................................................................................................... 12
X-Ray Crystallography .................................................................................................................................. 12
Immunoprecipitation ................................................................................................................................... 12
Radioimmunoassay ...................................................................................................................................... 12
Mass Spectrometry ...................................................................................................................................... 13
Enzyme-Linked Immunosorbent Assay (ELISA) ............................................................................................... 14
Indirect ELISA .......................................................................................................................................... 14
Sandwich ELISA........................................................................................................................................ 14
Edman Degradation ..................................................................................................................................... 15
Gram Staining .............................................................................................................................................. 15
Restriction Fragment Length Polymorphism (RFLP) ........................................................................................ 16
Salting Out and Dialysis ................................................................................................................................ 16
Reducing Sugar Tests ................................................................................................................................... 17
Tollen’s Test ............................................................................................................................................ 17
Benedict’s Test ........................................................................................................................................ 17
Fehling’s Test........................................................................................................................................... 17
cDNA Libraries ............................................................................................................................................. 18
 3

Gel Electrophoresis

Purpose: Separation of proteins, DNA, or RNA based on size and/or charge

Macromolecules (proteins, DNA, or RNA) of interest are placed in the lanes of a gel. For proteins and
small molecules of DNA and RNA, the gel will be polyacrylamide. For larger molecules of DNA (> 500 bp),
the gel will be agarose. An electrical charge is placed across the gel. At the bottom is the positively
charged anode and at the top is the negatively charged cathode. Keep in mind, since a voltage source is
applied to gel electrophoresis, it follows the same principles as an electrolytic cell. Negatively charged
molecules will travel toward the anode. Because of the resistance of the gel, larger molecules will have a
harder time moving and thus, the molecules will be separated by size with the smallest molecules
toward the bottom. The gel can then be stained for visualization, typically using Coomassie Blue dye. A
lane will be loaded with a collection of molecules of a known size, called a ladder, which can used to
determine the size of the molecules being ran. There are several different applications of gel
electrophoresis:

Native-PAGE
Native-PAGE is a polyacrylamide gel electrophoresis method for proteins that occurs under non-
denaturing conditions. This method will
separate proteins by size while retaining
their structure.

SDS-PAGE
SDS-PAGE is a polyacrylamide gel
electrophoresis method for proteins that
occurs under denaturing conditions to separate proteins by mass. Negatively-charged sodium dodecyl
sulfate (SDS) is added to the solution of proteins, denatures the proteins, and binds one SDS for every
two amino acids, giving all proteins the same charge-to-mass ratio. Since all proteins have the same
charge-to-mass ratio, they are separated solely on mass, with the smallest proteins found toward the
bottom of the gel. SDS will only interrupt non-covalent bonds, so if disulfide bridges are present in the
protein, they will not be broken. This is useful when analyzing proteins with multiple subunits.
 4

Reducing SDS-PAGE
Reducing SDS-PAGE is exactly like SDS-PAGE, but with the addition of a reducing agent, like β-
mercaptoethanol, which will reduce disulfide bridges and result in a completely denatured protein.

Isoelectric Focusing
Isoelectric focusing is a gel
electrophoresis method that separates
proteins on the basis of their relative
contents of acidic and basic residues. A
polyacrylamide gel with a pH gradient
(low pH on one side, high pH on the
other) is used. When proteins migrate
through the pH gradient gel, they will
travel toward the anode until the area
of the gel with the pH that matches their isoelectric point (pI). When a protein is at its pI, it has a net
charge of zero and will not be attracted to the positively charged anode so it will not move.

Western Blotting (Protein)

Purpose: Detection of a specific protein in a sample

Step 1: Proteins from a sample are loaded into an SDS-PAGE gel and separated based on size

Step 2: Proteins from the gel are transferred to a polymer sheet and exposed to a radiolabeled antibody
(sometimes using two antibodies; one specific to the protein of interest and another radiolabeled
antibody that binds to the first antibody) that is specific to our protein of interest

Step 3: The polymer sheet is viewed used autoradiography. The protein of interest that is bound to the
radiolabeled antibody will be visible.
 5

Southern (DNA) and Northern (RNA) Blotting

Purpose: Detection of a specific DNA (Southern blot) or RNA (Northern blot) sequence in a sample

Step 1: The DNA strand of interest is exposed to restriction enzymes that cut the DNA strand into
smaller fragments

Step 2: The newly cleaved strands of DNA are denatured using a solution of NaOH to create ssDNA
strands

Step 3: The single stranded cleaved strands of DNA undergo gel electrophoresis, separating them by
size. Smaller fragments will be found at the bottom of the gel. Polyacrylamide is used if the stands are
less than 500 base pairs. Agarose is used if the strands are over 500 base pairs.

Step 4: The DNA from the gel is transferred to a sheet of nitrocellulose paper and then exposed to a 32P
radiolabeled DNA probe that is complementary to our DNA of interest.

Step 5: The nitrocellulose paper is then viewed using autoradiography to identify the strand of interest.

NOTE: These methods are nearly identical for Southern and Northern blotting. The steps listed above
are for Southern blotting, however, the only difference is that Northern blotting uses RNA, so steps 1
and 2 are not done.
 6

DNA Sequencing (Sanger Dideoxynucleotide Sequencing)

Purpose: Used to determine the sequence of nucleotides in a strand of DNA

Modified nucleotides, known as “dideoxynucleotides” (ddNTPs), are used in this method. ddNTPs are
missing the OH group on the 3’ carbon, thus they are unable to create a new 5’→3’ phosphodiester
bond. This allows us to control the termination of replication.

Step 1: The DNA strand of interest is denatured using an NaOH solution to create a ssDNA strand that
we can use for replication

Step 2: The ssDNA strand of interest is added to a solution containing:

1. A radiolabeled DNA primer
that is complementary to the
gene of interest
2. DNA polymerase
3. All four dNTPs (dATP, dTTP,
dCTP, dGTP)
4. A very small quantity of a
single ddNTP (e.g., ddATP)
This step is done once for each of the
four nucleotides in separate solutions.

Step 3: Each solution containing a

specific dNTP and ddNTP are placed in
their own lane of a gel and ran under
gel electrophoresis. The gel is
transferred to a polymer sheet and
autoradiography is used to identify the
strands in the gel.

For each respective nucleotide, the insertion of a ddNTP will terminate replication and create various
strands of different length that correspond to that specific nucleotide. Therefore, the gel can be read
from bottom-to-top to determine the nucleotide sequence. The smaller the fragment, the further it
travels in the gel.
 7

Chromatography

Purpose: The separation of two or more molecules from a mixture

There are several different types of chromatography that can be used for separating or analyzing a
mixture of two or more molecules based on their properties. Traditionally, there are two components to
chromatography; a stationary phase, which is typically polar, and a mobile phase, which is typically non-
polar. Polar molecules are separated from a mixture by staying with the stationary phase, while non-
polar molecules stay with the mobile phase. However, if reverse-phase is specified, then the properties
of the two phases are switched. This also changes based on the type of
chromatography used, as some methods use ligands or gel beads.

Liquid Chromatography
In liquid chromatography, silica is traditionally used as the stationary
phase while toluene or another non-polar liquid is used as the mobile
phase.

High-Performance Liquid Chromatography (HPLC)

HPLC is a type of liquid chromatography that utilizes high pressures to
pass the solvent phase through a more finely-ground stationary phase,
which increases the interactions between the molecules and the
stationary phase, giving HPLC a higher resolving power. Molecules can
then be determined based on their absorbance and elution time as seen
on the right. In normal phase HPLC, stationary phase is polar, mobile
phase is nonpolar; in reverse phase HPLC, stationary is nonpolar, mobile is polar.

Gas Chromatography
Gas chromatography (also known as gas-liquid chromatography) is used to separate and analyze
molecules that can be vaporized. The mobile phase is an inert or unreactive gas, such as helium or
nitrogen, while the stationary phase is a thin layer of liquid or polymer that surrounds the walls of a
tube. The stationary phase allows more polar molecules to elute slower, giving them a higher retention
time.

Gel-Filtration (Size Exclusion) Chromatography

Gel-filtration chromatography (also known as size-exclusion
chromatography) is used to separate molecules by size rather than polarity.
Smaller molecules can enter the porous gel beads, allowing them to elute
later, while larger molecules that do not fit will elute faster. The gel beads
can be viewed as the stationary phase, while the solution in the column can
be viewed as the mobile phase.
 8

Ion-Exchange Chromatography
Ion-exchange chromatography will separate proteins by their net charge. The
column is filled with charged beads, either positive or negative. In cation-
exchange, negatively-charged beads are used which attract positively charged
proteins and negatively-charged proteins will elute first. In anion-exchange,
positively-charged beads are used which attract negatively-charged proteins
and positively-charged proteins will elute first.

Affinity Chromatography
Affinity chromatography will separate proteins based on their affinity
for a specific ligand. Beads that are bound to a specific ligand will be
used and proteins with a high affinity for that ligand will bind to the
beads, allowing proteins with a low affinity to elute first. The high
affinity proteins are then eluted by increasing the concentration of
the free ligand in the column, which competes for the active site of
the bound proteins.

Thin-Layer Chromatography
Thin-layer chromatography consists of a
small sheet of medium that is coated in an
adsorbent material, such as silica gel. The
polar silica is the stationary phase. The
molecules of interest are added to the
bottom of the sheet and the sheet is
placed in a non-polar liquid, such as
heptane, until it reaches the origin. The
mobile phase then travels up the plate
using capillary action, allowing the
molecules to move with it if they are
relatively non-polar. The spots are then
visualized using UV light.

The relative distances traveled between the molecules is represented by the Rf value, which is measured
as the ratio of the distance the molecule traveled from the origin to the distance the solvent front
traveled from the origin.
 9

Distillation

Purpose: Used to separate two or more molecules from a solution

Simple Distillation
Simple distillation is used to separate two molecules from a solution when their boiling points differ by
25o C or greater.

Fractional Distillation
Fractional distillation is used to separate two molecules from a solution when their boiling points differ
by less than 25o C.

Vacuum Distillation
Vacuum distillation is used to separate two molecules from a solution when their boiling points are high
and risk changing chemically.

Polymerase Chain Reaction

Purpose: Used to amplify a small quantity of DNA by several

orders of magnitude

Step 1: DNA strands and complementary DNA primers are heated

to 95o C for 15 seconds to separate the strands.

Step 2: The solution is abruptly cooled to 54o C to allow the

primers to anneal to each ssDNA.

Step 3: The solution is heated to 72o C and new complementary

strands are synthesized using Taq DNA polymerase.

Step 4: The cycle is repeated until the desired quantity of DNA is

synthesized.
 10

Spectroscopy

Purpose: Used for structural

determination of molecules

1
H-NMR Spectroscopy
The application of nuclear magnetic
resonance regarding the 1H isotope
within the molecules of a substance.

Chemical shift: The chemical shift on

the x-axis (δppm) represents the
amount of deshielding of electrons that
is caused by an adjacent heteroatom or
pi bond.
- 0 - 5 ppm → Alkane region
- 3 - 5 ppm → Alkane with a
heteroatom region
- 5 - 7 ppm → Alkene region
- 6 - 8 ppm → Aroma c region
- 9 - 10 ppm → Aldehyde region
- 10 - 13 ppm → Carboxylic acid region

Integration: The integration of the peak determines the number of equivalent hydrogens a signal
represents.

Neighbors: The number of peaks determines the number of neighboring hydrogens that are ≤ 3 bonds
away. The number of peaks equals the number of neighbors + 1.
- Singlet → No neighboring hydrogens
- Doublet → One neighboring hydrogen
- Triplet → Two neighboring hydrogens
- Quartet → Three neighboring hydrogens
- Quintet → Four neighboring hydrogens
- Sextet → Five neighboring hydrogens
- Septet → Six neighboring hydrogens
- Mul plet → Seven or more neighboring hydrogens
 11

13
C-NMR spectroscopy
The application of nuclear magnetic resonance regarding the 13C isotope within the molecules of a
substance

Chemical shift: The chemical shift on the x-axis (δppm) represents the amount of deshielding of
electrons that is caused by an adjacent heteroatom or pi bond.
- 0 - 70 ppm → Alkane region
- 90 - 120 ppm → Alkene region
- 110 - 160 ppm → Aroma c region
- 160 - 200 ppm → Carbonyl region

IR Spectroscopy
IR spectroscopy is a method that
is used to identify certain
functional groups within a
molecule. Only molecules that
have a dipole moment will show
absorbance. The x-axis is reported
in wavenumbers (reciprocal
centimeters) and the y-axis is
reported in percent absorbance.

Important regions:
- 1700 - 1750 → Carbonyls (sharp peak)
- 1720 - 1740 → Aldehydes
- 1700 - 1725 → Ketones
- 1735 - 1750 → Esters
- 1700 - 1725 → Carboxylic acids
- 3200 - 3600 → OH groups (broad peak)
- 3300 - 3400 → Amines
- The number of peaks are relative to the number of hydrogens on the amine (e.g., 1o
amines will have two peaks, 2o amines
will have one peak)

UV-Vis Spectroscopy
As the number of conjugated pi bonds increase, the
energy gap between the highest occupied molecular
orbital (HOMO) and lowest unoccupied molecular
orbital (LUMO) decreases, which means that light of a
lower energy is absorbed. This results in light with a
longer wavelength to be emitted (as seen on the right).
If a molecule absorbs green light, we see it as red.
 12

Autoradiography

Purpose: To visualize the location of a radioactive substance in a molecule or structure

In the scope of the MCAT, autoradiography is used primarily as an imaging technique to identify
radiolabeled atoms that are in a molecule, often as part of a Southern, Northern, or Western blot. The
radiolabeled substance is placed in contact with a photographic emulsion containing silver halide
crystals. The radiation of the radiolabeled substance converts the silver halide crystals into metallic
silver, producing an image.

X-Ray Crystallography

Purpose: To visualize the structure of a molecule, used often with proteins

In x-ray crystallography, a crystalline molecule causes a beam of incident x-rays to diffract into many
specific directions. A three-dimensional image can be constructed by measuring the different angles and
intensities of the diffracted rays.

Immunoprecipitation

Purpose: Used to purify proteins from a solution

In immunoprecipitation, a protein of interest can be precipitated from a solution by adding bead-

conjugated antibody that is specific to the protein of interest. The antibody is bound to some sort of
solid bead, which can be used for either extraction with a magnetic or by centrifuge.

Radioimmunoassay

Purpose: Used to determine the concentration of a protein of interest in a given sample

Similar to ELISA, the wells of a plate are coated with the primary antibody that is specific to our protein
of interest. Next, the radiolabeled protein, typically containing a tyrosine residue labeled with 125I, is
added to the wells and binds to the primary antibody. The concentration of that radiolabeled protein is
determined by counting the gamma emission. Next, a sample of the protein of interest in an unknown
concentration is added to the wells, where it competes for the active site on the primary antibody,
displacing an amount of the radiolabeled protein. The concentration of the radiolabeled protein is
determined again by counting the gamma emission. The difference in concentrations provides the
concentration of the protein of interest.
 13

Mass Spectrometry

Purpose: Used to determine the

molecular weight of a compound
and aid in determining the molecular
structure

In mass spectrometry, the sample is

vaporized and subjected to ionizing
conditions. The charged molecule
collides with an electron, resulting in
the ejection of an electron from the
molecule, making it a radical. The
charged radical can undergo
fragmentation or being detected.
The x-axis represents the
mass/charge ratio (m/z), which
essentially just means the molecules mass using only the lowest isotopes of the atoms involved (e.g.,
12
C, 1H, 35Cl). The y-axis represents the intensity, or relative abundance of the molecule, usually given as
a percentage.

Base peak: The tallest peak. This does not always represent the actual intact molecule, as it may
sometimes be a fragment of the molecule that is found in higher abundance.

Molecular ion peak (M): The peak that represents the molecule. The m/z value of this peak represents
the molecular weight of the molecule.

M+1 peak: The relative abundance of 13C in the molecule. Found in a relative abundance of 1.1%. So if
there is a M+1 with a m/z value of 4.4, it means that there are 4 carbons present (4.4/1.1 = 4).

M+2 peak: The relative abundance of either 37Cl or 81Br in the molecule. 37Cl will be found in a 3:1 ratio
relative to the M peak (e.g., if the M peak has a relative abundance of 90%, if the M+2 peak is at 30%, it
means there is chlorine present in the molecule). 81Br will be found in a 1:1 ratio relative to the M peak
(e.g., if the M peak has a relative abundance of 90%, if the M+2 peak is also at 90%, it means there is
bromine present in the molecule).

Fragments: There are three types of fragmentation that can occur, which can help identify the structure
of the molecule. 1) Alkane fragmentation, 2) alcohol dehydration, and 3) alpha cleavage. These are most
likely beyond the scope of the MCAT.
 14

Enzyme-Linked Immunosorbent Assay (ELISA)

Purpose: Used to identify the concentration

of a molecule of interest in a given sample

ELISA is an assay that uses primary antibodies

that are specific to a molecule of interest and
secondary antibodies that are specific to
primary antibodies and are conjugated with a
fluorophore, so their presence can be
measured via spectrophotometry. There are
two different methods of ELISAs that are
used.

Indirect ELISA
The molecule of interest (in this case, considered an antigen) is coated to the wells of a plate. A primary
antibody that is specific for that antigen is added and washed to remove any unbound antibodies. Next,
the secondary antibody is added. The secondary antibody is linked to an enzyme, often horseradish
peroxidase (HRP), that will be converted into a fluorophore when reacted with an oxidizing agent, such
as hydrogen peroxide. This will cause a change in color, which can be detected using
photospectrometry. The absorbance of the well is compared against a serial-diluted standard of known
concentration, and based on a determined curve using the standards, the concentration of the molecule
in the well can be determined.

Sandwich ELISA
The primary antibody that is specific to our molecule of interest is coated to the wells of a plate. Next,
the molecule of interest is added to the wells and then washed to remove any unbound molecules.
Next, the secondary antibody is added. The secondary antibody is linked to an enzyme, often
horseradish peroxidase (HRP), that will be converted into a fluorophore when reacted with an oxidizing
agent, such as hydrogen peroxide. This will cause a change in color, which can be detected using
photospectrometry. The absorbance of the well is compared against a serial-diluted standard of known
concentration, and based on a determined curve using the standards, the concentration of the molecule
in the well can be determined.
 15

Edman Degradation

Purpose: Used to sequence the amino acid

residues in a protein

Edman Degradation is conducted by removing

one amino acid residue at a time from the N-
terminus of the peptide. Phenyl isothiocyanate is
added to the N-terminus of a polypeptide, which
then cyclizes and breaks off, leaving an intact
polypeptide that is shortened by one residue. The
PTH-amino acid hybrid can then be analyzed
using chromatographic techniques to identify the
amino acid. This can be repeated until every
amino acid residue in the polypeptide is
identified. This method is limited in the fact that
it can only accurately identify polypeptides less
than 50 residues.

Gram Staining

Purpose: Used to differentiate bacteria into two groups, gram-positive or gram-negative, based on the
content of their cell wall

The sample of bacteria is heat-fixed to a slide and crystal violet dye is added. Iodide is added to the
bacteria, which binds to the crystal violet dye and traps it in the bacterial cell. The bacteria is then
washed with alcohol to remove any dye that was not taken up by the bacteria. Safranin is added as a
counterstain, so that the bacteria that did not stain with crystal violet can be visualized.

Gram-positive bacteria: Appears purple on the slide. Contains a thick peptidoglycan layer that takes up
the stain.
Gram-negative bacteria: Appears pink on the slide. Contains a thin peptidoglycan layer sandwiched
between two lipid bilayers that take up the safranin stain.
 16

Restriction Fragment Length Polymorphism (RFLP)

Purpose: Used to identify differences in homologous DNA sequences based on

the length of fragments caused by restriction enzymes

RFLP allows us to determine differences in two or more DNA sequences of the

same gene. Most often, this will be comparing a wild type (WT) gene with a
mutated version of the gene. The DNA strand is exposed to a restriction
enzyme, also called a restriction endonuclease. Restriction enzymes recognize a
specific palindromic sequence (the sequence is the same in the 5’ → 3’ direc on
in both strands) in the DNA and cleave both strands. Once the DNA sequence is
cleaved with the restriction enzymes, it can be ran through gel electrophoresis.
If a mutation has occurred, there may be a difference in the number and length
of strands between the WT and mutant genes, as the restriction points may
differ.

Salting Out and Dialysis

Purpose: Purification of proteins in a

solution

Adding salt to a solution containing

proteins can allow you to selectively
precipitate out proteins. Salting out is due
to the competition between the salt ions
and the protein for water to keep the
protein in solution. The salt concentration
at which a protein will precipitate varies
for each protein.

After a protein has been precipitated, the

salt can be removed via dialysis. The
solution is placed in a dialysis bag and
then added to a hypotonic solution. The
ions will pass through the semipermeable
membrane of the dialysis bag, while the
protein of interest is left inside.
 17

Reducing Sugar Tests

Purpose: Used to identify the presence of a reducing sugar in a

solution

A reducing sugar is any sugar that is capable of acting as a

reducing agent due to the presence of a free aldehyde or ketone
functional group. All monosaccharides are reducing sugars, since
they are capable of mutarotation, and therefore, will be found in
the open-chain form to some degree. However, not all
disaccharides, oligosaccharides, or polysaccharides are reducing
sugars, since some glycosidic bonds are between two anomeric
carbons (classified as a 1→2 bond), preven ng mutarota on.
Sucrose is an example of a non-reducing disaccharide while
maltose is an example of a reducing disaccharide (as seen in the
figure on the right). Certain reagents can test for the presence of a
free aldehyde or ketone group in order to identify the presence of
a reducing sugar.

Tollen’s Test
Tollen’s reagent tests for the presence of an aldehyde and can distinguish between aldoses and ketoses.
Ketones do not react unless they are α-hydroxy-ketones. A positive Tollen’s test is characterized by the
precipitation of elemental silver.

Tollen’s reagent consists of [Ag(NH3)2]NO3.

Benedict’s Test
Benedict’s reagent tests for the presence of an aldehyde. Ketones do not react unless they are α-
hydroxy-ketones. A positive Benedict’s test is characterized by a change in color from clear blue to brick-
red with the formation of a precipitate.

Benedict’s reagent consists of a mixture of sodium carbonate, sodium citrate, and copper (II) sulfate
pentahydrate.

Fehling’s Test
Fehling’s solution tests for the presence of an aldehyde. Ketones do not react unless they are α-hydroxy-
ketones. A positive Fehling’s test is characterized by a change in color from clear blue to brick-red with
the formation of a precipitate.

Fehling’s solution consists of two parts; Fehling’s A and Fehling’s B. Fehling’s A consists of aqueous
copper (II) sulfate. Fehling’s B consists of potassium sodium tartrate and sodium hydroxide.
 18

cDNA Libraries

Purpose: Used to create and house complimentary DNA (cDNA) strands that can be expressed in

bacterial vectors
In order to synthesize proteins for things like medication, such as insulin, we must clone the DNA
sequence for that protein in a bacterial vector. However, eukaryotes have the ability to undergo post-
transcriptional modification of hnRNA to create mRNA while prokaryotes do not. Therefore, we can not
insert eukaryotic DNA directly into a bacterial vector since it still contains introns. Instead, we must
synthesize a strand of cDNA from the protein of interest’s respective mRNA.

The mRNA transcript of the protein of interest is isolated and a complementary DNA primer containing
many thymine repeats and a free 3’-OH group is added, which anneals to the poly-A tail of the mRNA.
Next, the mRNA is exposed to reverse transcriptase and an excess of the four dNTPs so a
complementary cDNA strand can be synthesized, creating a cDNA-mRNA hybrid. The hybrid strand is
hydrolyzed using an alkaline solution. A primer is added to the new single-stranded cDNA and the
complementary strand is synthesized by DNA polymerase. The new double-stranded cDNA can be
inserted into a bacterial plasmid vector by using restriction enzymes and the bacteria will synthesize the
protein of interest.